Hematopoietic growth factors

ABSTRACT

A novel family of primate CSF-1-like polypeptides is provided via recombinant techniques.

The present invention relates to a novel family of CSF-1-likehematopoietic growth factors, and a process for producing them byrecombinant genetic engineering techniques.

BACKGROUND

Hematopoietins, i.e., hematopoietic growth factors, are proteins thatpromote the survival, growth and differentiation of hematopoietic cells.Colony stimulating factors (CSFs) are a subset of these hematopoieticgrowth factors tht are characterized by the ability to support thegrowth, in vitro, of colonies of hematopoietic cells arising fromprogenitor cells of bone marrow, fetal liver and other hematopoieticorgans.

The biochemical and biological identification and characterization ofcertain hematopoietins has been hampered by the small quantities of thenaturally occurring factors available from natural sources, e.g., bloodand urine. With recombinant genetic engineering techniques, however,some of these hematopoietins have been molecularly cloned,heterologously expressed and purified to homogeneity. [See D. Metcalf,"The Molecular Biology and Functions of the Granulocyte-MacrophageColony Stimulating Factors," Blood, 67(2):257-267 (1986).] Among thesehematopoietins are human and murine GM-CSF, human G-CSF, human CSF-1 andmurine IL3. Human IL-3 has also recently been identified [Y. C. Yang etal., Cell, 47(1):3-10 (1986)]. Both human GM-CSF [See, R. Donahue etal., Nature, 321:872-875 (1986)] and murine I L3 [See J. Kindler et al,Proc. Natl. Acad. Sci. U.S.A., 83:1001-1005 (1986)] have a demonstratedeffect on hematopoiesis in vivo.

A cDNA sequence for human urinary CSF-1 has been reported by E. S.Kawasaki, et al., Science, 230:291-196 (1985) [hereinafter "Kawasaki"],which, when expressed in a COS cell transient expression system,produced a 26 kD protein that competed with labeled murine CSF-1 in aradioreceptor assay. The protein also reportedly stimulated mouse bonemarrow proliferation, resulting in predominantly monocytic lineage typecolonies in the mouse bone marrow assay. The protein biological activitywas reportedly inhibited by neutralizing antisera for CSF-1.

BRIEF SUMMARY OF THE INVENTION

As one aspect of the invention, a family of CSF-1-like growth factorsare provided which are characterized by the amino acid sequences shownin FIG. 1, below. The amino acid sequences of the growth factors of thepresent invention are encoded by the DNA sequences also illustrated inFIG. 41. Similarly, DNA sequences which code for polypeptides coded forby the sequences of FIG. 1, but which differ in codon sequence due tothe degeneracies of the genetic code or differ in nucleotide sequencedue to cross-species variation or induced modifications also encode thenovel growth factors of this family described herein.

In addition to the DNA sequence homology to the sequences of FIG. 1, themembers of this novel family of growth factors are also characterized byhaving at least one biological property of a CSF-1-like growth factor.Preferably more than one CSF-1-like biological property is demonstratedby any one member of the family of growth factors of the presentinvention. "CSF-1-like biological property" is defined herein to includeone or more of the following biological characteristics and in vivo andin vitro activities One such property is the support of the growth anddifferentiation of progenitor cells committed to the monocyte lineages.For example, in a standard human bone marrow assay, a CSF-1-likebiological property is the stimulation of macrophage colonies. AnotherCSF-1-like biological property is complete inactivation bypre-incubation with 2-mercaptoethanol or an excess of rabbit antiserumraised to purified human urinary CSF-1. Additionally, CSF1-likeproperties include the ability to stimulate monocytes to produce otherCSF-like factors e.g. myeloid CSF, TNF and interfeon activity whichinteract with accessory and mature cells. Further CSF-1-like biologicalproperties are the stimulation of biological activities of maturemacrophages and the cells of continuous macrophage-like cell lines. Yetanother CSF-1-like biological property is an apparent molecular weightof about 50 to 90 kd by non-reducing sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE). Other biologicalproperties attributed to CSF-1 have been disclosed in the art.

As a further aspect of the present invention there are provided novelcDNA sequences coding on expression for CSF1-like polypeptides or growthfactors. These DNA sequences include those depicted in FIG. 1 in a 5' to3' direction and those sequences described above. Variations in the DNAsequences of FIG. 1 which are caused by point mutations or by inducedmodifications to enhance the activity or production of the polypeptidesare also encompassed in the invention. Similarly, synthetic polypeptideswhich wholly or partially duplicate continuous sequences of the aminoacid residues of FIG. 1 are also part of this invention. Thesesequences, by virtue of sharing primary, secondary or tertiarystructural and conformational characteristics with naturally-occurringCSF-1-like polypeptides of the invention may possess biological activityand/or immunological properties in common with the naturally-occurringproduct. Thus, they may be employed as biologically active orimmunological substitutes for naturally-occurring primate CSF-1-likepolypeptides in therapeutic and immunological processes.

As another aspect of the present invention, there is provided a novelmethod for producing the novel family of CSF-1-like growth factors. Themethod of the present invention involves culturing a suitable cell orcell line, which has been transformed with a vector containing a cDNAsequence coding on expression for a novel CSF-1-like polypeptide.Suitable cells or cell lines may be mammalian cells, such as Chinesehamster ovary cells (CHO). Another suitable mammalian cell line, whichis described in the accompanying examples, is the monkey COS-1 cellline. A similarly useful mammalian cell line is the CV-1 cell line. Alsosuitable for use in the present invention are bacterial cells. Forexample, the various strains of E. coli are well-known as host cells inthe field of biotechnology. Various strains of B. subtilis may also beemployed in this method. Many strains of yeast cells known to thoseskilled in the art are also available as host cells for expression ofthe polypeptides of the present invention. Additionally, where desired,insect cells may be utilized as host cells in the method of the presentinvention.

Another aspect of the present invention provides vectors for use in themethod of expression of these novel polypeptides. These vectors containthe novel DNA sequences described above which code for the novelpolypeptides of the invention. Alternatively, vectors incorporatingmodified sequences as described above are also embodiments of thepresent invention and useful in the production of these CSF-1-likepolypeptides. The vector employed in the method also contains selectedregulatory sequences in operative association with the DNA codingsequences of the invention and capable of directing the replication andexpression thereof in selected host cells. A variety of such regulatorysequences useful in the vectors of the present invention are well knownto those skilled in the art.

The members of the novel family of CSF-1-like growth factors may be usedin the treatment of diseases characterized by a decreased level ofhematopoietic cells, particularly those of myeloid, and moncytelineages. These factors may be used to directly stimulate monocyte andmacrophage production and may indirectly stimulate other hematopoieticlineages. Among conditions susceptible to treatment with thepolypeptides of the present invention is leukopenia, a reduction in thenumber of circulating leucocytes (white cells) in the peripheral blood.Leukopenia may be induced by exposure to certain viruses or toradiation. It is often a side effect of various forms of cancer therapy,e.g., exposure to chemotherapeutic drugs. Therapeutic treatment ofleukopenia with these CSF-1-like polypeptide compositions may avoidundesirable side effects caused by treatment with presently availabledrugs.

In addition these polypeptides may serve as activators of mature whitecells in cases of serious infection. These factors may be employed totreat infectious diseases characterized by intracellular parasitism,e.g., viral infections (herpes, cytomgalovirus), bacterial infections(Mycobacterium, Listeria), fungal infections (Candida) and parasiticinfections (Malaria) and the like.

Alone, or in combination with other hematopoietins, these factorsenhance macrophage function causing the activated macrophages to killtumor cells, to release alpha-interferon, to kill parasites or torelease and enhance other CSFs which may stimulate the proliferation andactivation of other blood cells.

The polypeptides of the present invention may also be employed, alone orin combination with other hematopoietins, in the treatment of otherblood cell deficiencies, including thrombocytopenia (plateletdeficiency), or anemia (red cell deficiency). Other uses for these novelpolypeptides are in the treatment of patients recovering from bonemarrow transplants, enhancing host defense during surgery and in burnpatients. These factors may also be employed to develop monoclonal andpolyclonal antibodies generated by standard methods for diagnostic ortherapeutic use.

Therefore, as yet another aspect of the invention are therapeuticcompositions for treating the conditions referred to above. Suchcompositions comprise a therapeutically effective amount of one or moreof the members of the family of CSF-1-like polypeptides of the presentinvention in admixture with a pharmaceutically acceptable carrier. Thiscomposition can be systematically administered either parenterally,intraveneously or subcutaneously. When systematically administered, thetherapeutic composition for use in this invention is, of course, in theform of a pyrogen-free, parenterally acceptable aqueous solution. Thepreparation of such a parenterally acceptable protein solution, havingdue regard to pH, isotonicity, stability and the like, is within theskill of the art.

The dosage regimen will be determined by the attending physicianconsidering various factors which modify the action of drugs, e.g. thecondition, body weight, sex, and diet of the patient, the severity ofany infection, time of administration and other clinical factors.Generally, the daily regimen should be in the range of 1-1000 microgramsof polypeptide or 50 to 5000 units (ie, a unit being the concentrationof polypeptide which leads to half maximal stimulation in a standardmurine bone marrow assay) of polypeptide per kilogram of body weight.This therapeutic composition may also be administered in conjunctionwith other human factors. A non-exclusive list of other appropriatehematopoietins, CSFs and interleukins for interaction with thepolypeptides of the present invention includes GM-CSF, G-CSF, Meg-CSF,erythropoietin (EPO), IL-1, IL-3, other CSF-1-like polypeptides, H-1,IL-4, IL-2, B-cell growth factor, B-cell differentiation factor andeosinophil differentiation factor. The dosage recited above would beadjusted to compensate for such additional components in the therapeuticcomposition. Progress of the treated patient can be monitored byperiodic assessment of the hematological profile, e.g. white cell countand the like.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a family of CSF-1-like growth factorscharacterized by amino acid sequences substantially homologous to theamino acid sequences illustrated in FIG. 1. These sequences may beencodedby the DNA sequences depicted in the Table or variously modifiedsequences as described above. These polypeptides are also characterizedby CSF-1-like biological properties.

The specific sequences illustrated in FIG. 1 are exemplary members ofthe growth factor family of the present invention. The 4 kb DNA of FIG.1 codes on expression for a novel CSF-1-like protein, named CSF-69. Itwas isolated from poly A+mRNA of the SV40 transformed trophoblast cellline TPA30-1 [ATCC #CRL-1583]. The sequence of FIG. 1 contains a longopen translational reading frame of 1662 nucleotides, encoding a 554amino acidpolypeptide. The protein coding region of the 4 kb sequenceextends from nucleotide #146 (methionine at -32) to nucleotide #1807which is followed by a TAG stop codon. There are four potentialasparagine-linked glycosylation sites illustrated by the characteristicsequences, Asn-X-Seror Asn-X-Thr. The remaining 2200 nucleotides of 3'non-coding sequence of the 4 kb region may have a regulatory role intranscription in the naturalhost. The 3' end of the sequence alsocontains an AT-rich segment includingseveral repeats of the sequenceATTTA, which is believed to be related to the RNA message stability[See, G. Shaw and R. Kamen, Cell, 46(5):659-677 (1986)].

This sequence contains three portions, i.e. from nucleotide #1 to #415,from nucleotide #419 to #689 and from nucleotide #1584 to #1823, whicharefound in the sequence of Kawasaki et al., supra. The coding region ofthe CSF-1 described by Kawasaki et al., differs from that describedherein by the deletion of 894 bp between nucleotides #689 and #1584recited in FIG. 1. ##STR1##

This approximately 4 kb DNA sequence is harbored in plasmid p3ACSF-69 inE.coli HB 101, which was deposited in the American Type CultureCollection, 12301 Parklawn Dr., Rockville, MD on Apr. 16, 1986 and givenaccession number ATCC 67092.

The 4 kb sequence of FIG. 1 codes on expression for one novel CSF-1-likeprotein of the present invention, CSF-69. CSF-69 is characterized by anapparent molecular weight of approximately 70-90 kD when analyzed bypolyacrylamide gel electrophoresis under non-reducing conditions.However,if this analysis is performed after reduction of the CSF-69, theprotein ischaracterized by an apparent molecular weight of 35-45 kDsuggesting that CSF-69 is a disulfide linked homo-dimer of 35-45 kDsubunits. In p3ACSF-69conditioned media, CSF-69 demonstrated CSF-1-likeactivity in in vitro mouse and human bone marrow assays.

The approximately 61 kD precursor encoded by the sequence of FIG. 1 isprocessed at the amino terminus by removal of a 32 residue signalpeptide and in the carboxy terminal region by removal of about 333residues to yield a subunit of approximately 189 amino acids with apredicted molecular weight of 21 kD. Thus the mature CSF-1 monomer hasGlu at its amino terminal and extends at least through to amino acid Leuat position #189 (See FIG. 1). This subunit retains two of the fourpotential sites for addition of asparagine-linked carboyhydriate thatare present in the sequence of FIG. 1. Glycosylation of the 21 kDpolypeptide at these two positions is presumed to account for most ofthe remaining mass of the 35-45 kD subunit of CSF-69.

Other novel CSF-1-like proteins of the present invention are encoded byonly a portion of the sequence of FIG. 1. One such CSF-1-like protein isencoded by nucleotides 1 to 1332 of the cDNA sequence of FIG. 1 and hasactivity, upon expression, in the murine bone marrow assay describedbelow. Similarly, cDNA sequences of from nucleotides 1 to 699, fromnucleotides 1 to 881 and from nucleotides 1 to 1012, when blunted andinserted into the expression vector described below also produced activeprotein. Additionally, another full length cDNA clone had a cytosine atposition 1678 of FIG. 1, rather than a thymidine. This change conservedthe amino acid, alanine, in the sequence.

The family of CSF-1-like growth factors provided herein also includesfactors encoded by the sequences of FIG. 1 into which nucleotidemodifications have been deliberately engineered. Such modifications intheDNA sequences can be made by one skilled in the at using varioustechniques. Specific modifications of interest in these CSF-1-likerelatedsequences include the replacement of one or more of the cysteineresidues in the coding sequences with other amino acids, e.g. serine, toeliminate a disulfide bridge. The 699 nucleotide factor, for example,has eliminatedseveral of the cysteines entirely. Mutagenic techniquesfor such replacement are well known to one skilled in the art. [See,e.g., U.S. Pat. No. 4,518,584.]

Other specific mutations of the sequences of the CSF-1-like factorsdescribed herein involve modifications of one or both of theglycosylationsites. The absence of glycosylation or only partialglycosylation results from amino acid substitution at one or both of theasparagine-linked glycosylation recognition sites present in thesequences of the CSF-1-likefactors shown in FIG. 1. These tripeptidesequences are either asparagine-X-threonine or asparagine-X-serine,where X is usually any amino acid. A variety of amino acid substitutionsat one or more of the three amino acid positions of a glycosylationrecognition site, especiallythe first and/or third such positions,result in non-glycosylation at the modified tripeptide sequence. By wayof example, Asn₁₂₂ of the sequence of FIG. 1 can be replaced withglutamine in one such modified CSF-1-like factor. The resulting factor(Gln₁₂₂) should contain only one asparagine-linked carbohydrate moietyrather than two such moieties. Those skilled in the art will appreciatethat analogous glycoproteins having the same monoglycosylation may beprepared by substituting another amino acid at position 122, and/or bysubstituting other amino acids at the other positions within theglycosylation recognition sites, e.g., inserting valine at Thr₁₂₄.Similarly, the Asn at position 122 and/orThr at position 124 may bealtered by a mutagenic technique to other amino acids to deglycosylatethe factor at that site. Alternatively, both of thesites may be alteredas above. [See, e.g. A. Miyajima et al., EMBO J., 5(6):1993-1197(1986)].

The following examples illustrate the method of the present inventionemploying the DNA sequence of FIG. 1 to produce CSF-69.

EXAMPLE I Construction of an exemplary mammalian expression vectorp3ACSF-69

To construct a mammalian vector for expression of a novel CSF-1-likeprotein, the cDNA sequence depicted in FIG. 1 above was adapted withrestriction endonuclease enzyme XhoI linkers (New England Biolabs) andligated into XhoI-digested, phosphatased COS cell expression vector pXM.pXM contains the SV40 enhancer, major adenovirus late promoter, DHFRcoding sequence, SV40 late message poly A addition site and VaI gene.pXM further contains a linker sequence with restriction endonucleasesites forKpnI, PstI and XhoI. The plasmid resulting from the XhoIdigestion of pXM and the insertion of the linker and the XoI adapted DNAsequence of FIG. 1coding for a CSF-like protein was designatedp3ACSF-69. p3ACSF-69 (ATCC #67092) can be transformed by conventionaltechniques into a suitable mammalian host cell for expression of theCSF-like protein. Exemplary hostcells are mammalian cells and celllines, particularly primate cell lines, rodent cell lines and the like.

A similar expression vector may also be prepared containing the other,CSF-1-like sequences identified above, or containing only the amino acidcoding regions of those sequences of FIG. 1, with the 5' and 3'non-codingregions deleted. One skilled in the art can construct othermammalian expression vectors comparable to p3ACSF-69 by cutting the DNAsequence of FIG. 1 from the plasmid with XhoI and employing well-knownrecombinant genetic engineering techniques and other known vectors, suchas pCD [Okayama et al., Mol. Cell Biol. 2:161-170 (1982)] and pJL3, pJL4[Gough et al., EMBO J. 4:645-653 (1985)]. The transformation of thesevectors into appropriate host cells can result in expression of aCSF-1-like protein.

Similarly, one skilled in the art could manipulate the CSF-1-likesequencesby eliminating or replacing the mammalian regulatory sequencesflanking thecoding sequence with yeast, bacterial or insect sequences tocreate non-mammalian vectors. Thus this sequence would then beexpressable in yeast, bacterial or insect host cells. For example, thecoding sequence ofFIG. 1 could be cut from p3ACSF-69 with Xhol andfurther manipulated (e.g.,ligated to other known linkers or modified bydeleting non-coding sequencestherefrom or altering nucleotides thereinby other known techniques). The modified CSF coding sequence could thenbe inserted into, for example, a known bacterial vector using proceduressuch as described in T. Taniguchi et al, Proc. Natl. Acad. Sci U.S.A.,77:5230-5233 (1980). This exemplary bacterial vector could then betransformed into bacterial host cells and the CSF-69 protein expressedthereby.

Similar manipulations can be performed for the construction of an insectvector [See, e.g., procedures described in published European patentapplication 155,476] or a yeast vector [See, e.g., procedures describedinpublished PCT application WO 86 00639] for expression of theCSF-1-like proteins in insect or yeast cells.

EXAMPLE II Expression of a CSF-1-like Protein

Plasmid DNA, prepared from E. coli HB101 containing p3ACSF-69 asdescribed in Maniatis et al., Molecular Cloning, A Laboratory Manual,Cold Spring Harbor Laboratory, (1982) was purified by conventionalmethods involving equilibrium centrifugation in cesium chloridegradients containing ethidium bromide COS cells (ATCC CRL 1650) weretransfected with the purified DNA at a concentration of approximately 5ug plasmid DNA per 106 COS cells and treated with chloroquine accordingto the procedures described in G. G. Wong et al., Science, 280:810-815(1985) and R. J. Kaufman et al., Mol. Cell Biol., 2:1304 (1982). 72hours following transfection, p3ACSF-69 medium can be harvestedcontaining a protein whichdemonstrates CSF-1-like activity in standardbone marrow assays, as discussed in Example III below.

EXAMPLE III CSF Activity in In Vitro Assays

A. Mouse assay

Mouse bone marrow assays were conducted as described in D. Metcalf, TheHemopoietic Colony Stimulating Factors, Elsevier Press, New York (1984)with the following modifications:

(a) 2×10⁵ bone marrow cells per ml were employed in the assay;

(b) final assay volume was 100 ul; and

(c) assays were set up in standard 96 well microtitre plates.

Bone marrow was obtained from the femurs of 6-25 week old female Balb/cmice (Jackson). Using WEHI 3 conditioned medium [J. C. Lee et al., J.Immunol., 128:2393-2398 (1982)]which contains mouse L cell conditionedmedium as a standard control, one dilution unit of activity was definedasthat concentration of protein which results in a maximal response inthis bone marrow assay, i.e., approximately 15 to 20 colonies per 2×10⁴mouse bone marrow cells.

p3ACSF-69 conditioned medium was found to be active to at least 1:104dilution in a mouse bone assay and produced mainly monocytic lineagetype colonies. The number and type of cells in a maximal response willvary with the strain and age of the mouse donors.

B. Human Assay

Human bone marrow assays, employing non-adherent bone marrow cells, wereperformed as described in G. G. Wong, et al, supra. p3ACSF-69conditioned medium was active to a 1:50 dilution in human bone marrowassays and produced predominantly monocytic lineage-type colonies.

EXAMPLE IV Construction of CHO cell lines expressing hiqh levels ofCSF-69

One method for producing high levels of the CSF-1-like polypeptides frommammalian cells involves the construction of cells containing multiplecopies of the heterologous CSF-1-like gene. The heterologous gene can belinked to an amplifiable marker, e.g., the dihydrofolate reductase(DHFR) gene for which cells containing increased gene copies can beselected by propagation in increasing concentrations of methotrexate(MTX) according to the procedures of Kaufman & Sharp, J. Mol. Biol.,supra. This approach can be employed with a number of different celltypes.

p3aMCSF R1 #1-69 and the DHFR expression plasmid pAdD26SV (A)3 (Kaufman& Sharp, Mol. Cell Biol., supra) were co-transfected into DHFR-deficientCHOcells, DUKX-BII, by calcium phosphate coprecipitation andtransfection. Theinitial DHFR expressing transformants were selected forgrowth in alpha media with dialyzed fetal calf serum, and subsequentlyselected for amplification by growth in increasing concentrations of MTXas described in Kaufman, et al., Mol. Cell Biol. 5:1750 (1983).

One clone, designated 5/9 mα3-18 (0.2) which was selected for growth in0.2 micromolar MTX was found to express high levels of biologicallyactive CSF-69. This cell line consistently generated conditioned mediumwhich was active in supporting murine macrophage colony formation at a1:60,000 final dilution. These cells (one 10 cm dish) as well as theparent CHO cells were labeled with 1 mCi of ³⁵ S-met (NEN) in 4 mlofMinimal Essential Medium (MEM) for 4 hours at 37° C. The resultingconditioned media samples were incubated with antiserum raised inrabbits with purified urinary CSF-1. The antigen-antibody complexes wereprecipitated by adsorbtion to Staphylococcus aureus cells (Cal Biochem).The complexes were solubilized in a loading buffer lacking reducingagent according to U. K. Laemmli, Nature, 227: 680-685 (1970). To reducesamplesthey were brought to 100 mM 2-mercaptoethanol and incubated at37° C. for 30 minutes. Following electrophoresis in 10 % polyacrylamidegel, the pattern of labeled proteins were visualized by fluorography(Enhance, NEN) using Kodak XAR film.

Analysis of these immunoprecipitates by SDS polyacrylamide gelelectrophoresis under non-reducing conditions revealed that theconditioned medium from the CSF-1-like protein producing CHO cellscontained two heterogeneous CSF-1-like protein species of apparent sizes70-90 kD and greater than 150 kD. The observed size heterogeneity oftheseCSF-1-like proteins is typical of many glycoproteins. Analysis ofthe same samples following reduction revealed that the mobility of the70-90 kD species of CSF-1-like protein shifted to a position consistentwith a molecular weight of 35-45 kD while the relative mobility of thelarger species (greater than 150 kD) was unaffected by the treatment.Thus at least two different CSF-1-like proteins are expressed by the 5/9mα3-18 (0.2) cells: a 70-90 kD protein comprising a disulfidelinkeddimer of a 35-45 kD subunit, and a much larger species.

EXAMPLE V Purification of CSF-69

The CHO-cell conditioned media containing 0.5% fetal bovine serum andDMEM-F12 is diluted 1:1 with water. The diluted media is then applied toaQAE `Zeta-Prep` cartridge (LKB) which is equilibrated in 40 mM Tris pH7.4.The flow through containing unbound protein was discarded. Boundprotein was washed with 40 mM Tris, pH 7.4 and eluted with 40 mM Tris,pH 7.4 and 0.75M NaCl. The eluate is then diluted with water to aconcentration of 0.5M NaCl. Tween 20 was added to 0.05% and this mixtureloaded at approximately 1 column volume/hour on to a lentil lectin.Sepharose 4B column [Pharmacia] which had been equilibrated in 20 mMTris, pH 7.4, 0.5MNaCl and 0.05% Tween 20. The column was washed with2-5 cv, 20 mM Tris, Ph 7.4, and 0.5 M NaCl. Specifically-bound proteinwas eluted with 20 mM Tris, 0.2M alpha methylmannopyranoside, 0.5M NaCland 0.05% Tween 20, and then acidified with 10% trifluoroacetic acid[TFA]. The eluate was subjected to reverse phase liquid chromatographyon a column equilibrated in 30% acetonitrile and 0.1% TFA. Protein waseluted with ascending acetonitrile in 0.1% TFA. Protein collectedbetween 45 and 50% acetonitrile was neutralized in tubes with Tris, pH8.5 and analyzed.

The preliminary analysis of CSF-69 reveals a specific activity ofapproximately 10⁶ bone marrow units per milligram [see bone marrow assayin Example III].

Numerous modifications and variations in practice of this invention areexpected to occur to those skilled in the art upon consideration of theforegoing descriptions of preferred embodiments thereof. Suchmodifications and variations are believed to be encompassed in theappended claims.

What is claimed is:
 1. A DNA sequence consisting essentially of a DNAcoding on expression for a polypeptide capable of stimulatingproliferation of monocytic cells in both humans and mouse bone marrowassays and having a peptide sequence encompassing the mature form of thepolypeptide of FIG. 1 and allelic variations thereof also capable ofstimulating proliferation of monocylic cells in both human and mousebone marrow assays.
 2. A transformation vector comprising a DNA sequenceaccording to claim 9 in operative association with an expression controlsequence therefor.
 3. The vector according to claim 10, which isp3ACSF-69.
 4. A host cell containing the vector of claim 10 and beingcapable of producing said polypeptide.